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Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping
Author(s) -
Loo Rachel R. Ogorzalek,
Mitchell Charles,
Stevenson Tracy I.,
Martin Stephen A.,
Hines Wade M.,
Juhasz Peter,
Patterson Dale H.,
Peltier John M.,
Loo Joseph A.,
Andrews Philip C.
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180312
Subject(s) - chromatography , isoelectric focusing , chemistry , mass spectrometry , bovine serum albumin , polyacrylamide gel electrophoresis , polyacrylamide , gel electrophoresis , silver stain , proteome , matrix (chemical analysis) , analytical chemistry (journal) , sodium dodecyl sulfate , matrix assisted laser desorption/ionization , desorption , biochemistry , adsorption , microbiology and biotechnology , biology , polymer chemistry , enzyme , organic chemistry
Matrix‐assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin‐layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of α‐ and β‐chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one‐dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time‐lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa α‐subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)‐polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.